THE ELECTRON-TRANSFER REACTIONS OF NADPH-CYTOCHROME P450 REDUCTASE WITH NONPHYSIOLOGICAL OXIDANTS

The steady-state kinetics of oxidation of rat liver NADPH:cytochrome P 450 reductase (EC 1.6.2.4) by quinones, aromatic nitrocompounds, ferri cyanide, Fe(EDTA)(-), and cytochrome c has been studied. The logarithm s fo bimolecular rate constants of reduction (k(cat)/K-m) of quinones and nitrocompounds increase with the increase in their single-electron reduction potential (E(7)(1)), reaching a maximum value at E(7)(1) > - 0.15 V. The reactivities of nitroaromatics are about by an order of ma gnitude lower than the reactivities of quinones. For a series of nitro aromatics including the compounds with previously undetermined E(7)(1) values, an orthogonality was found between their reactivities toward cytochrome P450 reductase, flavocytochrome b(2) (EC 1.1.2.3), and the NADPH:adrenodoxin reductase (EC 1.18.1.2)-adrenodoxin system. This ind icates the absence of significant specific interactions during these r eactions. The effects of ionic strength on reaction kinetics and the c haracter of inhibition by a product of reaction, NADP(+), are in accor dance with the reduction of oxidants at the negatively charged site in the surroundings of FMN of P450 reductase. Quinones inactivate oxidiz ed reductase modifying the NADP(H) binding site. The redox cycling of quinones markedly slows the inactivation. The kinetic data presented a re consistent with an outer-sphere electron transfer mechanism. The an alysis of kinetics of reduction of cytochrome c, ferricyanide, and Fe( EDTA)(-) using the model of Mauk et al. (A. G. Mauk, R. A. Scott, and H. B. Gray (1980) J. Am. Chem. Soc. 102, 4360-4363) gives calculated d istances of FMN from the surface of protein globule, 0.33-0.63 nm. The data from nitroreductase reactions of cytochrome P450 reductase, flav ocytochrome b(2), and adrenodoxin were used for approximate evaluation of previously unknown E(7)(1) of nitrocompounds. (C) 1994 Academic Pr ess, Inc.